PROJECT SUMMARY/ABSTRACT The ability for cells to detect and respond to metabolic cues is critical to maintaining homeostasis, and perturbations in the sensing mechanisms that respond to oscillations in metabolic flux are the root cause of many diseases, including sepsis, autoimmunity, cancer, and diabetes. There is mounting evidence that protein post- translational modifications (PTMs) are the critical sensors for these metabolic fluctuations and are often dysregulated in disease. Currently, we have a fundamental gap in our understanding of the composition, abundance, and enzymatic control of PTMs and how they are altered in disease. My laboratory focuses on the identification and characterization of PTMs and how they are regulated in both health and disease. To accomplish this goal, we have developed sensitive methods to identify and quantify global changes in PTMs across a broad spectrum of biological samples. Using this approach, we have identified a novel lysine PTM that is derived from a glycolytic by-product. These PTMs are elevated when glyoxalase 2 (GLO2) is inhibited, resulting in reduced glycolytic output and disrupted one-carbon metabolism. Our primary goal is to establish the therapeutic efficacy of a GLO2 inhibition strategy for the treatment of metabolic disorders. My research program is dedicated to understanding four fundamental questions: 1) How does GLO2 control one-carbon metabolism and cellular redox? GLO2 knockout cells have reduced glutathione and increased oxidative stress. We will quantify the role of GLO2 in the regulation of de novo glutathione synthesis. In addition, the role of GLO2 in the regulation of antioxidant responses will be evaluated in a cellular model for oxidative stress and inflammatory signaling. 2) How are LactoylLys modifications regulated? We will employ quantitative proteomics using CRISPR-Cas9 knockout cell lines of candidate proteins to identify enzymatic regulators of LactoylLys modifications in cells. 3) Is GLO2 a viable target for the treatment of glycolysis- dependent disease states? A xenograft model will be employed using GLO2 knockout cell lines to quantify proliferation and metabolic regulation in vivo. This will determine the therapeutic feasibility of targeting GLO2 for the treatment of disease. 4) Are LactoylLys modifications functional histone marks? We have identified histones as targets for modification by LactoylLys modifications in unstimulated cells. The presence of these PTMs basally suggests a putative role in transcriptional regulation. We will use proteomics to identify site-specific modifications and putative ?reader? domains for LactoylLys modifications in cells. Our primary goal is to establish the role of GLO2 and LactoylLys modifications in cell metabolism and chromatin biology. This project will address a fundamental gap in our basic understanding of how cell metabolism is regulated. Understanding how these PTMs regulate homeostasis is a critical first step to understanding their role in disease. Due to the far-reaching implications of this project and the broad applications for the treatment of highly glycolytic disease states, this research program is an ideal fit for the ESI MIRA Award.